It is a continuing challenge to develop anti-cancer agents that are capable of inhibiting the growth of, or killing, cancer cells, without affecting normal cells. Researchers have focused on genetic mutations in cancer cells to find clues to discover such new anti-cancer drugs.
Many cancer cells have mutations in genes involved in the G1 cell cycle arrest checkpoint. Such genes include impaired tumor suppressor genes, e.g., p53, Rb, p16INK4, and p19ARF. Alternatively, such mutations can cause expression of oncogenes, e.g., MDM-2 and cyclin D. In addition to these, excessive growth factor signaling can be caused by the over expression of growth factors. Together with these gain-of-function mutations, growth factor receptors or downstream signal-transducing molecules can cause cell transformation by overriding the G1 checkpoint. In contrast, few cancers have disrupted G2 cell cycle arrest checkpoints. Thus, the G2 checkpoint is usually retained in cancer cells with the impaired G1 checkpoint.
If the G2 checkpoint could be selectively disrupted, cancer cells with an impaired G1 checkpoint would become more sensitive to DNA-damaging treatment, as compared to normal cells (with intact G1), since progression through G1 and G2 without repairing such damage induces apoptosis.
The mechanism that promotes the cell cycle G2 arrest after DNA damage is conserved among species from yeast to human. In the presence of damaged DNA, Cdc2/Cyclin B kinase is kept inactive because of inhibitory phosphorylation of threonine-14 and tyrosine-15 residues on Cdc2 kinase. At the onset of mitosis, the dual phosphatase Cdc25 kinase removes these inhibitory phosphates and thereby activates Cdc2/Cyclin B kinase.
In fission yeast, the protein kinase Chk1 is required for the cell cycle arrest in response to damaged DNA. Chk1 kinase acts downstream of several rad gene products and is modified by the phosphorylation upon DNA damage. The kinases Rad53 of budding yeast and Cds1 of fission yeast are known to conduct signals from unreplicated DNA. It appears that there is some redundancy between Chk1 and Cds1 because elimination of both Chk1 and Cds1 was culminated in disruption of the G2 arrest induced by damaged DNA. Interestingly, both Chk1 and Cds1 phosphorylate Cdc25 kinase and promote Rad24 binding to Cdc25, which sequesters Cdc25 to cytosol and prevents Cdc2/Cyclin B activation. Therefore Cdc25 appears to be a common target of theses kinases and presumably an indispensable factor in the G2 checkpoint.
In humans, both hChk1, a human homologue of fission yeast Chk1, and Chk2/HuCds1, a human homologue of the budding yeast Rad53 and fission yeast Cds1, phosphorylate Cdc25C at serine-216, a critical regulatory site, in response to DNA damage. This phosphorylation creates a binding site for small acidic proteins 14-3-3s, human homologues of Rad24 and Rad25 of fission yeast (Lopez-Girona (1999) Nature 397:172-175). The regulatory role of this phosphorylation was clearly indicated by the fact that substitution of serine-216 to alanine on Cdc25C disrupted cell cycle G2 arrest in human cells (Peng (1997) Science 277:1501-1505).